Classifying disease stage and elucidating a prognosis, which allow the most effective medical intervention, are unattainable when the underlying pathological changes, or the correlates of stage and prognosis, are associated with heterogeneous cells states within a larger population. Single cell analyses can provide an unsurpassed means to measure and unravel heterogeneity in complex biological systems, and thereby to understand the basis for (or to identify correlates of) changes in biological function and disease processes. Here we propose to develop a rapid assay for detection of antigen-specific responses in single T cells from small amounts of blood, based on state-of-the-art techniques that are sensitive and robust. Our proposal aims at developing a test that provides tuberculosis (TB) diagnosticians with the critical ability to distinguish stable latent Mycobacterium tuberculosis infection (when the asymptomatic subject is not progressing to disease, is not infectious, and does not require treatment) from preclinical disease (when the asymptomatic subject is developing disease, is still not infectious, and requires early treatment to block progression of disease and drastically curb transmission of infection). Our multidisciplinary team includes expertise in development of novel single cell analysis methodology, cellular immunology, and biomarker research for TB, which still causes millions of cases of disease and death worldwide every year. Our assay is expected to yield multi-parameter measurements of single T cell functional states by integrating (i) use of artificial Ag-presenting cells (aAPC) to activate T cell receptor signaling and stimulation of gene expression, with (ii) measurement of inducible tell-tale markers of T cell activation and function by quantitative flow cytometry. Induced gene expression will be detected by mRNA enumeration using single molecule fluorescence in situ hybridization (smFISH). The research plan is articulated in four aims, each focused on the development of a specific aspect of the assay: (1) read-out: detection of activation markers in single T cells by smFISH and flow cytometry following conventional stimulation; (2) stimulation: response to aAPC assessed by detection of activation markers in single T cells; (3) response to infection-stage-specific Ag: association of single T cell responses with disease vs asymptomatic infection; (4) infection-stage-specific functional T cell signatures: multi-parameter characterization of single T cell responses and association with disease vs asymptomatic infection. The proposed plan should lead to recognizing and treating active TB prior to the appearance of microbiological and clinical signs and symptoms of disease. This is the current holy grail in TB diagnosis as it is considered to be critical to TB elimination efforts. The new assay principles will be translatable for diagnosis and disease staging of any pathology with T cell involvement, including other infectious diseases, cancer, autoimmunity, and transplantation. PUBLIC HEALTH RELEVANCE: Successful intervention in many diseases is often severely limited by insufficient understanding of the complex underlying pathology. In particular, the ability to classify the disease stage and to elucidate a prognosis may be poor or even impossible when pathological changes are identifiable only in a small number of cells within a larger population. Here we propose to develop a new immunological test that can identify, from among persons carrying an asymptomatic infection with Mycobacterium tuberculosis (the bacteria that cause tuberculosis), those individuals progressing toward disease and becoming infectious. Two billion people (about one-third of the world's population) are currently infected with the TB bacteria, with the highest concentration of new cases in resource-poor areas of South-East Asia and sub-Saharan Africa. Even though 90-95% of infected persons do not become sick, the number of infected individuals is high enough to give rise to 8 million new cases of TB and almost 2 million deaths each year worldwide. Existing blood-based clinical immunodiagnostic assays recognize TB infection but are not geared to distinguish between asymptomatic infection and active disease. The latter is currently identified only when patients excrete TB bacilli while coughing or sneezing (it is estimated that, by the time of diagnosis, a person with active TB has already infected up to twenty contacts). Thus, for public health purposes, it is critical to diagnose active disease when the patient is still asymptomatic and non-infectious. We propose to develop a new, rapid test requiring small amounts of blood that detects tell-tale immune cell subpopulations distinguishing the relevant groups. The detection method in this test requires basic flow cytometry, a technique routinely utilized in the clinical diagnosis of immunopathologies and fully accessible to district/peripheral level clinical laboratories that currently operate a fluorescence microscope in resource-poor regions. Translation of the proposed diagnostic methodology to clinical practice will allow identification and treatment of early cases of TB, thus impeding transmission and helping eliminate TB. While the present proposal is directed to a specific set of end-users in TB clinics, the proposed assay has broad public health relevance, because the same principles and methodologies are directly applicable to any infectious and non-infectious disease that can be characterized based on the properties of single immune cells involved in the body's recognition of or response to a particular condition.